Squaraine dyes

ABSTRACT

Squaraine dyes and compositions of matter containing such dyes are disclosed. The squaraine dyes have an absorption maximum greater than 600 nanometers and are particulary useful in conjunction with a helium/neon (He/Ne) laser. Some of the squaraine dyes are hydrophilic and are therefore water soluble or water compatible and others of the squaraine dyes are lipophilic.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to squaraine dyes and compositions of mattercontaining such squaraine dyes. Squaraine dyes have a structural elementof cyclobutenolate, generally being a condensation product of squaricacid (dihydroxycyclobutenedione) and an active compound such as apyrrole or an aniline. Squaraine dyes have been found to have a numberof uses based on their unique properties. One such use involves layeredphoto responsive imaging devices. Such devices are responsive to visiblelight and infrared illumination needed for laser printing.

Many of the known squaraine dyes are fluorescent compounds absorbinglight in the wavelength region of from about 400 to about 1000nanometers. Consequently, another use that has been proposed forsquaraine dyes is in the area of assays. Fluorescent compounds haveachieved wide application in assays because of their ability to emitlight upon excitation with energy within certain energy ranges. Byvirtue of this ability, fluorescers have found employment as labels inchemical or biological processes. Various compounds can be conjugated tofluorescent compounds, the conjugate subjected to some type ofpartitioning, and the fate of the conjugate determined by irradiatingthe sample with light and detecting the zone in which the conjugateexists. Fluorescent labels find use in immunoassays, involving specificbinding pairs, such as ligands and receptors, for example, antigens andantibodies. For such use, the fluorescent compounds preferably aresoluble in an aqueous medium or at least water compatible.

Another use of fluorescent compounds is to incorporate such compoundinto a cell wall or a liposome. The cell or the liposome with thefluorescent compound incorporated therein can also be employed inassays. For example, dyes incorporated into cell membranes are useful inthe area of blood typing where a change in fluorescence as a result ofagglutination of cells is determined. Liposomes containing fluorescentdyes also find use in the assay area, particularly in immunoassays. Forthis purpose it is desirable that the fluorescent dyes be lipophilic.

Laser beams find use in the assay area as means for irradiating afluorescent compound. In the field of assays it is important to avoidcontributions to a signal produced in relation to the amount of analyte,which contributions result from other than the analyte. For example,serum or plasma from a patient is often used to conduct the assay. Serumis itself fluorescent. The materials that cause fluorescent in serum orplasma normally absorb light at a wavelength below 600 nanometers.Therefore, it is desirable to employ fluorescent dyes in fluorescentassays that have an absorption maximum greater than 600 nanometers. Itis also desirable to employ a laser beam to provide the source of energyfor activation of the fluorescent dye. One such laser is the helium/neonlaser which emits light at 633 nanometers.

2. Description of the Related Art

Various squarate dyes are discussed by Sprenger, et at., Angew. Chem.,80, 541 (1968); Sprenger, et al., Angew. Chem., 79, 581, 1967; Sprenger,et al., Angew. Chem. Internat. Edit., 5:894, 1966; and Maaks, et al.,ibid., 5:888, 1966.

Novel unsymmetrical squaraine systems are disclosed in U.S. Pat. No.4,521,621. A process for the preparation of squaraine compositions inthe presence of an amine component is disclosed in U.S. Pat. No.4,524,220. A process for preparation of squaraine compositions isdisclosed in U.S. Pat. No. 4,525,592. A process for preparation ofsquaraine compositions from half esters of squaric acid is disclosed inU.S. Pat. No. 4,524,219. Processes for the preparation of squarainecompositions are described in U.S. Pat. No. 4,524,218. A process for therecovery of high purity squaric acid is described in U.S. Pat. No.4,523,980. Amine derivatives of squaric acid are described in U.S. Pat.Nos. 4,123,270, 4,353,971, 3,838,095, and 3,824,099.

SUMMARY OF THE INVENTION

The present invention concerns novel compounds that are squaraine dyesand novel compositions containing such dyes. The novel squaraine dyes ofthe invention have an absorption maximum greater than 600 nanometers.Some of the squaraine dyes are hydrophilic and therefore watercompatible or water soluble. Other squaraine dyes of the invention arelipophilic.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Generally, the novel squaraine dyes of the invention have the followingformula. ##STR1## wherein:

D is independently selected from the group consisting of: ##STR2##

Y is independently selected from the group consisting of: ##STR3##

Z in D and Z in Y are independently selected from the group consistingof carbon, nitrogen, oxygen, sulfur, and selenium;

s is 2 when Z is carbon, 1 when Z is nitrogen, and 0 when Z is oxygen,sulfur, or selenium;

R is independently hydrogen or a substituent having from 1 to 30 atomsother than hydrogen, which atoms are selected from the group consistingof carbon, oxygen, nitrogen, sulfur, halogen or atomic number 9 to 53,boron with the proviso that, where D and Y are the same and all but oneR group is lower alkyl (1 to 4 carbon atoms), the remaining R group hasat least one heteroatom such as, for example, oxygen, nitrogen, orsulfur, or is a chain of a least five carbon atoms which may or may nothave such heteroatom; R may be taken together with one or more other Rgroups to form one or more rings, usually five or six membered rings; Rmay contain one or more groups which are hydroxy, carboxy, includingesters and amides thereof, sulfonic acid, amine including primary,secondary, and tertiary amine, aryl including phenyl, carbamate,succinimidyl and the like.

Preferred compounds of the present invention have the following formula:##STR4## wherein:

R₁ and R₄ are independently selected from the group consisting of loweralkyl (1 to 4 carbon atoms);

R₂ and R₃ are independently selected from the group consisting of:

(a) lower alkyl (1 to 4 carbon atoms),

(b) alkyl of from 5 to 20 carbon atoms, preferably 10 to 16 carbonatoms, including cycloalkyl, and ##STR5## wherein

R₁₆ is hydroxyl, or

lower alkoxy (1 to 4 carbon atoms), or

--NH₂ NH₂ and salts thereof, or

--NH(CH₂)_(c) NH₂ and salts thereof, c is 1 to 5, or ##STR6## d is 1 to5, or ##STR7## b is 1 to 5, or

--NH(CH₂)_(t) --S--S--CH₃

wherein t is 1 to 5, or ##STR8## wherein R₂₉ is ##STR9## wherein R₃₀ islower alkyl (1 to 4 carbon atoms), or ##STR10## wherein k is 1 to 5, orlower alkoxy (1 to 4 carbon atoms), or ##STR11## and salts thereof, ordioleoylphosphotidylethanolamine, or --NH(CH₂)_(n) NHR₁₇ wherein R₁₇ is##STR12## wherein R₁₈ is lower alkyl (1 to 4 carbon atoms); --(CH₂)_(r)NH₂ wherein r is 1 to 5; hydrogen including acid salts thereof;##STR13## and n is 1 to 5, preferably 2 to 3, or --NH(CH₂)_(p) --R₁₉wherein R₁₉ is ##STR14## wherein q is 1 to 5; --NH₂ ; phenyl, aminosubstituted phenyl; and p is 1 to 15, preferably 2 to 12, and

R₅ and R₆ are independently selected from the group consisting ofhydrogen, hydroxyl, lower alkoxyl (1 to 4 carbon atoms), alkoxyl of from5 to 20 carbon atoms, ##STR15## wherein R₂₀ is hydrogen or lower alkoxyl(1 to 4 carbon atoms); with the proviso that only one of R₂ or R₃ islower alkyl (1 to 4 carbon atoms) when R₅ and R₆ are both hydroxyl orboth hydrogen; and ##STR16## wherein R₉ is hydrogen, lower alkyl (1 to 4carbon atoms) of --CH₂ (CH₂)_(w) SO₃ H wherein w is 1 to 5, preferably 2to 3; and ##STR17## wherein

Z is independently ##STR18## or S;

R₁₂, R₁₃, R₁₄, and R₁₅ are independently hydrogen, lower alkyl (1 to 4carbon atoms) or R₁₂, R₁₃ and R₁₄, R₁₅ are taken together to form##STR19##

R₁₀ and R₁₁ are independently selected from the group consisting of:

hydrogen,

lower alkyl (1 to 4 carbon atoms),

alkyl of from 5 to 20 carbon atoms, including cycloalkyl, ##STR20##

wherein z is 0 to 5, ##STR21##

wherein R₂₁ is hydrogen, or lower alkyl (1 to 4 carbon atoms) and v is 0to 5, and ##STR22## and

β₁ and β₂ are independently hydrogen, halogen (chlorine, bromine,fluorine), or lower alkoxy (1 to 4 carbon atoms), with the proviso thatonly one of β₁ or β₂ is hydrogen or halogen when R₁₀ and R₁₁ are bothhydrogen or lower alkyl of 1 or 2 carbon atoms; and ##STR23## wherein:

R₂₂, R₂₃, and R₂₅ are independently hydrogen, lower alkyl (1 to 4 carbonatoms), or alkyl of from 5 to 20, preferably 10 to 20, carbon atoms, and

R₂₄, R₂₆, R₂₇, and R₂₈ are independently hydrogen, lower alkyl (1 to 4carbon atoms), alkyl of from 5 to 20, preferably 10 to 16, carbon atoms,hydroxyl, lower alkoxyl (1 to 4 carbon atoms), or alkoxyl of from 5 to20, preferably 10 to 16, carbon atoms.

Particularly preferred compounds of the present invention have thefollowing formulas: ##STR24## wherein:

(a) R'₁, R'₂, R'₃, and R'₄ are all

(i) --CH₂ CH₂ CH₃,

(ii) --CH₂ (CH₂)₁₄ CH₃, ##STR25##

(b) R'₁ and R'₄ are both --CH₂ CH₃ and R'₂ and R'₃ are both

(i') --CH₂ (CH₂)₄ CH₃, ##STR26##

(c) R'₁, R'₂, and R'₃ are all --CH₂ CH₃ and R'₄ is ##STR27##

(d) R'₁ and R'₂ are --CH(CH₂)₂ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is##STR28##

(e) R'₁ and R'₂ are --CH₂ (CH₂)₁₄ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is##STR29##

(f) R'₁ and R'₂ are --CH₂ (CH₂)₂ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is##STR30## or

(g) R'₁ and R'₂ are --CH₂ (CH₂)₁₄ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is##STR31## and ##STR32## wherein R"₁, R"₂, R"₃ and R"₄ are all --CH₂(CH₂)₁₄ CH₃ ; and ##STR33## wherein:

(a) R"₇ and R"₈ are --CH₂ (CH₂)₁₄ CH₃ ;

(b) R"₇ is hydrogen and R"₈ is --CH₃ ;

(c) R"₇ is hydrogen and R₈ is ##STR34## or

(d) R"₇ is hydrogen and R"₈ is ##STR35## and ##STR36## wherein:

(a) R₁₀ and R₁₁ are both:

(i) --CH₂ (CH₂)₂ CH₃,

(ii) --CH₂ (CH₂)₄ CH₃,

(iii) --CH₂ (CH₂)₅ CH₃,

(iv) --CH₂ (CH₂)₆ CH₃,

(v) --CH₂ (CH₂)₁₀ CH₃,

(iv) --CH₂ (CH₂)₁₄ CH₃, ##STR37## wherein:

(a) R'"₁₀ and R'"₁₁ are --CH₂ (CH₂)₂ CH₃ and β₁ and β₂ are hydrogen; or

(b) R'"₁₀ and R'"₁₁ are --CH₂ (CH₂)₁₄ CH₃ and β₁ and β₂ are hydrogen; or

(c) R'"₁₀ and R'"₁₁ are --CH₂ CH₃ and β₁ and β₂ are --OCH₂ CH₃ ; and##STR38## wherein R'₉ is hydrogen or (b) --CH₂ CH₂ CH₂ SO₃ H

It will be appreciated by those skilled in the art that those compoundsmentioned above which have a polar or polar groups such as hydroxyl,amine, carboxy, sulfonic acid and the like will be hydrophilic or atleast water compatible. Furthermore, it will be appreciated by thoseskilled in the art that those compounds mentioned above that lack suchpolar groups will tend to be lipophilic in character.

The compounds of the present invention can be prepared by a reactionsequence, some or all of the individual steps of which are separatelyknown in the art. Some of the squaraine dyes of the present inventioncan be made according to procedures similar to those described bySprenger et al., Angew. Chem., 80, 541 (1968); Springer et al., Angew.Chem., 79, 581 (1967); Sprenger et al., Angew. Chem., Internat. Edit.,5, 894 (1966); and Maaks et al., Angew. Chem. Internat. Edit., 5, 888(1966). In general, squaric acid (dihydroxycyclobutenedione) iscondensed with an active compound such as a pyrrole or an aniline. Thecondensation is conducted under conditions for removing water from thereaction mixture. For example, the condensation can be carried out underreflux in an alkanol/benzene solvent mixture. The resulting product canbe collected and purified by, for example, recrystallization,distillation, chromatography, or the like. The group or functionalityimparting hydrophilicity or lipophilicity to a particular compound ofthe invention can be part of an initial reactant for the condensation orit can be introduced after the condensation by conventional techniques.

The squaraine dyes of the invention can be conjugated to specificbinding pair (sbp) members such as antigens and antibodies by techniquesthat are known in the art. Such conjugation can be the result of directbond formation between the squaraine dye and the sbp member. On theother hand, a linking group as described above can be introduced intothe squaraine dye or the sbp member for attachment to the othercomponent. A functionality for attachment such as carboxylic acid,hydroxyl, thio, amino, aldehydic, amido, activated ethylenes such asmaleimide, sulfonic acids, and the like can be introduced into thesquarate dye or the sbp member if such functionality is not originallypresent in the dye or the sbp member. Methods of conjugation involvingsbp members are described in, e.g., U.S. Pat. No. 3,817,837, thedisclosure of which is incorporated herein by reference.

Some of the compounds of the invention have properties that are verydesirable for their use in assays. The compounds have a high extinctioncoefficient, a high quantum efficiency, approaching one, chemicalstability, and satisfactory Stokes shift. Furthermore, where thecompounds are to be used in the presence of serum or other composition,which is in itself fluorescent, the compounds absorb energy in asubstantially different range from that absorbed by the other compoundsin the medium. As mentioned above, the present compounds have anabsorption maximum greater than 600 nanometers.

Some of the compounds of the invention are useful because of theirlipophilic character. For example, lilophilic squaraine dyes of theinvention may be incorporated into a cell and thereby cause the cell tobecome fluorescent. Because of their lipophilic character some of thesquaraine dyes may be employed in the formation of liposomes wherein thelipophilic material is incorporated into the liposome.

Generally the molar extinction coefficient for the novel squaraine dyesat the wavelength of the exciting light is greater than 1,000 and oftengreater than 10,000 per mole per centimeter. The novel squaraine dyeshave a high quantum yield, normally greater than 0.05 and often greaterthen 0.03.

EXAMPLES

The invention is further demonstrated by the following illustrativeexamples.

EXAMPLE 1 PREPARATION OF XIII

Twenty-five (25) ml of 50% n-butanol in benzene was dried by azeotropicdistillation for thirty minutes under a nitrogen atmosphere. One mmol(165 mg) of N,N-diethyl-3-aminophenol, 1 mmol (114 mg) of3,4-dihydroxy-3-cyclobutene-1,2-dione, and 1 mmol (315 mg) ofN-ethyl-2,3,3-trimethylindolenium iodide were added and the reaction wasrefluxed under nitrogen for three hours with azeotropic removal ofwater. The reaction rapidly turned from a golden yellow color to green,then to deep blue. At the end of the reaction all solvents wereevaporated off and the crude blue product was chromatographed on silicagel 60, using a gradient of methanol in chloroform to effect separation.24 mg (6%) yield) of pure blue product was obtained (λ max(DMSO):642nm).

EXAMPLE 2 PREPARATION OF VI(a)(iv)

1,3-bis[4-N,N-dicarboxymethylamino)-2hydroxyphenyl]-2,4-dihydroxycyclobuteneliydiumdihydroxide, bis (inner salt) tetra tert-butyl ester.

Twenty-five (25) ml of 50% n-butanol in benzene was dried by azeotropicdistillation for thirty minutes under a nitrogen atmosphere; 1.18 mmol(400 mg) of 3-dicarboxymethylaminophenol di-tert-butyl ester and 0.59mmol (68 mg) of 3,4-dihydroxy-3-cyclobutene-1,2-dione were added. Thereaction mixture was refluxed under nitrogen for five hours withazeotropic removal of water. The reaction rapidly turned from a goldenyellow color to light green, then to an increasingly deeper blue-green.The product gradually precipitated from solution. After the reactionmixture cooled to room temperature, the deep blue-green solid productwas filtered off and dried in vacuo. The reaction solvent was evaporatedoff and the remainder of the product was purified by preparative thinlayer chromatography on silica gel 60 using CHCl₃ as a developingsolvent. A total of 303 mg (68% yield) or pure compound was obtained (λmax = 642 nm).

EXAMPLE 3 PREPARATION OF VI(a)(v)

Sixty-six (66)μ mol (50 mg) of compound VI(a)(iv) was dissolved in 10 mlof dry methylene chloride; 2 ml of dry trifluoroacetic acid was added.The reaction mixture was stirred at room temperature under nitrogen for2.5 hours. The product precipitated out of solution and was filtered offand dried in vacuo. 32 mg (91% yield) of product was obtained, pure bythin layer chromatography and high performance liquid chromatograph (λmax=634 nm in methanol).

EXAMPLE 4 PREPARATION OF VIII(b), VIII(c) and VIII(d)

1-[4-(diethylamino)-2-hydroxyphenyl]-3-[4-(diethylamino)-2-alkoxyphenyl]-2,4-dihydroxycy dihydroxide, bis (inner salt)

These compounds were all prepared by the same method:

0.1 mmol (41 mg) of1,3-bis[4-(diethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis(inner salt) (DEAS) was reacted with 0.4 mmol of theappropriate alkyl halide (methyl iodide, t-butylbromoacetate, orbromoacetic acid) in the presence of 0.2 mmol of potassium tert-butoxidein 5 ml of dry dimethylformamide at room temperature under nitrogen for15 hours. The reaction was worked up by pouring the deep blue DMFsolution into 10 ml of water, extracting with 2×20 ml of ethyl acetate,washing the ethyl acetate phases with 1×20 ml of saturated brine, andcombining and drying the organic phases over sodium sulfate. Followingpreparative thin layer chromatography on silica gel GF, using 10%methanol in chloroform as developing solvent, the desired product wasobtained pure. Compound VIII (b) was obtained in 13% yield (λmax=652nm). Compound VIII (c) was obtained in 24% yield. Compound VIII(d) was obtained in 25% yield (λ max=647nm).

EXAMPLE 5 PREPARATION OF IX(a)(xiv)

Twenty-five (25) ml of 50% n-butanol-toluene was dried by azeotropicdistillation under a nitrogen atmosphere and 1 mmol (252 mg) ofchloroindoline [prepared by the reaction of 2,3,3-trimethylindoleninewith epichlorohydrin, neat, 100° C.] and 0.5 mmol (57 mg) of3,4-dihydroxy-3-cyclobutene-1,2-dione were added. The reaction mixturewas heated at reflux under nitrogen for two hours with azeotropicremoval of water. The reaction rapidly turned from golden color to greento deep blue. The product crystallized out of the hot solution. Afterallowing the reaction to cool to room temperature, the pure product wasfiltered off; 152 mg (52% yield) of a pure green-blue solid wasobtained. (λ max (DMSO):646 nm)

EXAMPLE 6 PREPARATION OF IX(a)(xv)

Twenty (20) ml of 50% n-butanol in benzene was dried by azeotropicdistillation for thirty minutes under nitrogen and 0.35 mmol (40 mg) of3,4-dihydroxy-3-cyclobutene-1,2-dione and 0.70 mmol (292 mg) of theindoline disulfide [prepared by the reaction of the chloroindoline ofExample 5 with dithioerythritol according to standard prodecures] wereadded. The reaction mixture was refluxed under nitrogen for two hoursunder nitrogen with azeotropic removal of water. The reaction rapidlyturned from a yellow color to deep green, and then gradually to deepblue. An additional 0.1 mmol of 3,4-dihydroxy-3-cyclobutene-1,2-dionewas added after the first hour. The reaction was evaporated to drynessin vacuo, then purified by preparative thin layer chromatography onsilica gel GF, using 5% methanol in chloroform as a developing solvent.The product was chromatographed a second time using 10% methanol inchloroform as developing solvent. Following extraction of the productfrom silica gel with chloroform and methanol and evaporation ofsolvents, 216 mg (68% yield) of pure product was obtained as a bluecrystalline solid with a reddish sheen (λ max (aq. DMSO):641 nm, ε221,000).

EXAMPLE 7 PREPARATION OF VI(c)(ii)

1-[4-(diethylamino)-2-hydroxyphenyl]-3-[4-N-ethylcarboxymethylamino)-2-hydroxyphenyl]-2,4-dihdihydroxide, bis (inner salt) [VI(c)(ii)] was prepared by refluxing withazeotropic water removal equimolar amounts of 3-diethylamino phenol,squaric acid, and N-ethyl-N-3-hydroxyphenyl glycine methyl esterprepared as in Example 6 in 2:1 (v/v) n-butanol: benzene followed byisolation of the monomethylester-dye [VI(c)(i)] by silical gelchromatography. The deep blue methyl ester was hydrolyzed with NaOH togive DECAS [VI(c)(ii)] after acidification.

The NHS (N-hydroxysuccinimide) ester of DECAS [VI(c)(iii)] was generatedby addition of 1.3 mg of EDAC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to asolution of 3.0 mg of DECAS and 0.99 mg of NHS in 120 μl of freshlydistilled (CaH₂) DMF (dimethylformamide) of 0°. Tlc (silica, 90:10 v/vchloroform: methanol) indicated the presence of the NHS ester of DECASand disappearance of starting material after 1 hr.

EXAMPLE 8 PREPARATION OF VI(c)(xiii)

to 2.78 mg of acid VI(c)(ii) from Example 7 in 120 μl of drydimethylformamide was added 1.51 mg of sulfo-NHS (Pierce) followed by1.27 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.The stoppered reaction was stirred by means of a magnetic stir bar. Thinlayer chromatography (silica, 90:10 chloroform: methanol vol/vol) showedthe gradual disappearance of the blue spot corresponding to VI(c)(ii)and the appearance of a new blue spot (R_(f) =0.2). After 4 hr reactionwas judged to be complete. The sulfo-NHS ester VI(c)(iii) was used insolution without further isolation.

EXAMPLE 9 PREPARATION OF XII(c)

5-ethoxy-2-methylbenzthiazole was alkylated in toluene at 65° with ethyliodide. The resulting salt was condensed with squaric acid at reflux intoluene/butanol with azeotropic removal of H₂ O to give a blue solutioncontaining XII(c). Purification on silica tlc (thin layerchromatography) eluted with 95:5 CH₂ Cl₂ :CH₃ OH gave XII(c) as a bluesolid. Fluorescence in 0.01M sodium phosphate, 0.15M NaCl, 0.005M NaN₃,0.01M β-cyclodextrin, pH7: λ max/ex=651, λ max/em=664 nm.

EXAMPLE 10 PREPARATION OF X

5-methoxy-2,3,3-trimethylindolenine was prepared by condensation of4-methoxyaniline and 3-hydroxy-3-methyl-2-butanone in 2:1 xylene:butanolat reflux with ZnCl₂ catalysis. To 240 mg of5-methoxy-2,3,3-trimethylindolenine in 10 ml of toluene and 30 ml ofn-butanol was added 70 mg of squaric acid. The mixture was refluxed 6 hrwith azeotropic removal of H₂ O. Concentration of the blue solution gave220 mg of X as green crystals: MS (EI) 456 (M⁺). Fluorescence in DMSOcontaining 0.001M HCl: λ max/ex=650, λ max/em=664.

EXAMPLE 11 PREPARATION OF VI(c)(x)

Condensation of acid VI(c)(ii) from Example 7 (0.27 mmol) andm-aminophenylboronic acid (0.27 mmol) with excess1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (≧0.3 mmol)in dry DMF at 5° over 2 days after silica tlc eluted with 90:10 CH₂ Cl₂:CH₃ OH gave 10 mg of VI(c)(x) having a fluorescence emission maximum of664 nm when excited >600 nm in DMF.

EXAMPLE 12 PREPARATION OF VI(c)(vi)

To 4.8 mg of amino dye VI(c)(iv) in 2 ml of dry THF in a round-bottomedflask containing a magnetic stir bar was added 1.3 mg of ethylchlorofumarate (prepared by the method of U. Eisner, G. Elvidge, and A.Linstead, J. Chem. Soc., 1501 (1951)). Then 1.4 μl of triethylamine wasadded with stirring. Thin layer chromatography (silica, 90:10toluene:methanol v/v) showed the gradual development of a new blue spotand decrease of starting VI(c)(iv). After overnight stirring, thereaction mixture was purified by preparative thin layer chromatography(tlc) (silica, 90:10 toluene:methanol). The major moving blue band wasscraped from the plate and eluted from the silica with methanol.Evaporation of the solvent gave 1 mg of blue residue which was a singlespot by thin layer chromatography (as above) and identified as VI(c)(vi)by 300 mHz nmr in CDCl₃ with TMS as standard.

EXAMPLE 13 PREPARATION OF VI(c)(iv)

To a solution of 0.27 mmol of VI(c(iii) in 12 ml of drydimethylformamide (DMF) at 0° was added dropwise 19.5 mg ofethylenediamine in 1 ml of DMF. A green solution resulted. After 1 hr ofstirring at 0° the reaction mixture was stripped of DMF under vacuum.The residue was washed with water and centrifuged. The washing andcentrifugation was repeated once. The Rf on silica was 0.23 forVI(c)(iii) and 0.68 for VI(c)(iv) when eluted with 90:10 CH₂ Cl₂ :CH₃ OH(v/v). The crude solid was further purified on preparative tlc elutedwith 90:10 CH₂ Cl₂ :CH₃ OH to give 10 mg of VI (c)(iv).

EXAMPLE 14 PREPARATION OF VI(c)(vii)

Acid VI(c)(ii) from Example 7 (219 mg) was dissolved in dry DMF at 0°.N-hydroxy-succinimide (63 mg) was added followed with stirring by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg).After 30 min at 0° followed by 23° for 4 hr tlc (silica, 90:10 CH₂ Cl₂:CH₃ OH) showed a new blue spot, Rf=0.94 and absence of the blue spotfor 34, Rf=0.13. To the presumed NHS ester was added a solution of 74 mgof h₂ NCH₂ CH₂ SSCH₃ in DMF at 0°. After overnight stirring 0°-5°, tlc(silica, 80:20 ethylacetate:hexane) showed absence of NHS ester. The DMFwas removed under vacuum, and the residue was purified by tlc (silica,ethylacetate:hexane 80:20) to give 35 mg of blue solid.

EXAMPLE 15 PREPARATION OF VI(c)(ix)

To a solution of 0.27 mmol of VI(c)(iii) in 12 ml of DMF at 0° was added31 mg of diethylenetriamine dropwise with stirring. After stirring 1 hr,tlc (silica, 90:10 CH₂ Cl₂ :CH₃ OH) showed absence of VI(c)(iii)(Rf>0.09) and presence of a non-moving blue spot Rf.sup.˜ 0. The DMF wasthen removed under vacuum and the crude blue solid was purified by tlc(silica CH₃ OH:CH₂ Cl₂ 15:85).

    ______________________________________                                        EXAMPLE 16                                                                    Fluorescence Data                                                                                     max      max                                          Compound   Solvent      γ ex                                                                             γ em                                                                         φ                                   ______________________________________                                        VI(a)(i)   DMF          652      666                                          VI(a)(ii)  toluene      621      632                                          VI(a)(v)   methanol     635      652  0.79                                               PBS          637      658  0.26                                               PBS/βCD 653      665  0.                                                 PBS/SDS      636      658  0.23                                    VI(b)(i)   toluene      632      648                                                     methanol     628      648                                                     PBS/βCD 642      656                                          VI(b)(iv)  methanol     632      651                                                     water        632      656                                          VI(b)(vi)  methanol     632      65l                                                     water        634      659                                          VI(b)(vii) methanol     632      652                                                     water        636      655                                          VI(b)(ix)  methanol     632      652                                                     water        632      652                                          VI(b)(xiv) metanol      631      652                                                     water        629      650                                                     PBS/βCD 647      660                                          VII        toluene      634      650                                          VI(c)(vii) toluene      638      653                                                     DMF,acid     648      663                                                     PBS/βCD 648      662                                                     PBS/triton   648      661                                          VI(c)(x)   DMF                   664                                          VI(f)      water        636      662                                                     methanol/CH.sub.2 Cl.sub.2                                                                 646      664                                                     PBS/5% cholate                                                                             648      665                                          VI(g)      CHCl.sub.3   640      655                                                     DMF, acid    648      665  0.86                                               PBS triton   647      663  0.95                                               PBS/tween    643      658                                                     methanol                   0.82                                    VIII(a)    toluene      636      654                                                     CHCl.sub.3   636      654                                          VIII(b)    PBS/βCD 652      664  1.0                                     VIII(d)    methanol     633      650  0.20                                               PBS/βCD 648      660  0.25                                               PBS          639      659  0.02                                    XIV(a)     toluene      669      679                                                     DMF          674      690                                                     CHCl.sub.3   660      680                                                     PBS/βCD 676      692                                                     PBS/γCD                                                                              676      692                                          XIV(b)     methanol/water                                                                             666      672                                          IX(a)(i)   methanol     628      639  0.11                                               DMF          640      656                                                     PBS/SDS      634      644  0.36                                               red cell     641      651                                          IX(a)(ii)  methanol     628      639  0.10                                               PBS/SDS      633      643  0.36                                    IX(a)(vi)  toluene      641      658                                                     ethanol      635      645                                                     methanol     629      640  0.l8                                               DMF          640      652                                                     PBS/SDS      636      645  0.48                                    IX(a)(xiii)                                                                              PBS/βCD 630      638                                                     PBS/SDS      634      645                                                     PBS/CTAB     639      646                                          IX(b)      PBS/βCD 629      641                                                     PBS/CTAB     638      648                                          X          DMSO/acid    683      704                                                     PBS/βCD 650      664                                          XI         methanol     650      670  0.1                                     XIII       DMSO         642      662                                                     PBS/βCD 647      659  0.28                                               PBS/SDS      636      653  0.71                                    XII(a)     DMF          674      690                                          XII(b)     toluene      682      694  0.34                                    XII(c)     PBS/βCD 651      664                                          ______________________________________                                    

"DMF/acid" means DMF acidified with 5μl/ml of 1N Hcl. "DMSO" meansdimethylsulfoxide. "DMSO/acid"" means DMSO acidified with 5 μl/ml of 1NHCl. "PBS" means phosphate buffered saline (10 mM sodium phosphate, 5 mMNaN₃, 0.15 N NaCl, pH7. "PBS/βCD" means PBS containing 0.01Mμ-cyclodextrin. "PBS/SDS" means PBS containing 10 mg/ml of sodiumdodecyl sulfate. "PBS/CTAB" means PBS containing 10 mg/ml of cetyltrimetylammonium bromide. "PBS/γCD" means PBS containing 0.01Mγ-cyclodextrin. "PBS/Triton" means PBS containing 10 mg/ml ofTriton-X-100. "PBS/Tween" means PBS containing 10 mg/ml of Tween-20. "φ"means fluorescence quantum yield. "λ_(ex) ^(max) " means wavelength ofmaximum excitation in the fluorescence spectrum. "λ_(em) ^(max) " meanswavelength of maximum emission in the fluorescense spectrum.Fluorescense spectra were measured on a Perkin Elmer 650-40 FluorescenceSpectrophotometer.

The invention has been described in detail with particular reference tothe above embodiments. It will be understood, however, that variationand modifications can be effected within the spirit and scope of theinvention.

What is claimed is:
 1. A compound of the formula: ##STR39## wherein: R₁and R₄ are independently selected from the group consisting of loweralkyl (1 to 4 carbon atoms);R₂ and R₃ are independently selected fromthe group consisting of: (a) lower alkyl (1 to 4 carbon atoms), (b)alkyl of from 5 to 20 carbon atoms, and ##STR40## wherein R₁₆ ishydroxyl, of lower alkoxy (1 to 4 carbon atoms), or --NH₂ NH₂ and saltsthereof, or --NH(CH₂)_(c) NH₂ and salts thereof, c is 1 to 5, or##STR41## d is 1 to 5, or ##STR42## b is 1 to 5, or --NH(CH₂)_(t)--S--S--CH₃ wherein t is 1 to 5, or ##STR43## wherein R₂₉ is ##STR44##wherein R₃₀ is lower alkyl (1 to 4 carbon atoms), or ##STR45## wherein kis 1 to 5, or lower alkoxy (1 to 4 carbon atoms), or ##STR46## and saltsthereof, or dioleoylphosphotidylethanolamine, or --NH(CH₂)_(n) NHR₁₇wherein R₁₇ is ##STR47## wherein R₁₈ is lower alkyl (1 to 4 carbonatoms); --(CH₂)_(r) NH₂ wherein r is 1 to 5; hydrogen including acidsalts thereof; ##STR48## and n is 1 to 5, preferably 2 to 3,--NH(CH₂)_(p--R) ₁₉ wherein R₁₉ is ##STR49## wherein q is 1 to 5; --NH₂; phenyl, amino substituted phenyl; and p is 1 to 15, preferably 2 to12, and R₅ and R₆ are independently selected from the group consistingof hydrogen, hydroxyl, lower alkoxyl (1 to 4 carbon atoms), alkoxyl offrom 5 to 20 carbon atoms, ##STR50## wherein R₂₀ is hydrogen or loweralkoxyl (1 to 4 carbon atoms); with the proviso that only one of R₂ orR₃ is lower alkyl (1 to 4 carbon atoms) when R₅ and R₆ are both hydroxylor both hydrogen.
 2. A compound of the formula: ##STR51## wherein: (a)R'₁, R'₂, R'₃, and R'₄ are all(i) --CH₂ CH₂ CH₃, (ii) --CH₂ CH₂ CH₂ CH₃,(iii) --CH₂ (CH₂)₁₄ CH₃, ##STR52## (b) R'₁ and R'₄ are both --CH₂ CH₃and R'₂ and R'₃ are both --CH₂ (CH₂)₄ CH₃ ; ##STR53## (c) R'₁, R'₂, andR'₃ are all --CH₂ CH₃ and R'₄ is: ##STR54## (d) R'₁ and R'₂ are--CH(CH₂)₂ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is ##STR55## or (e) R'₁ andR'₂ are --CH₂ (CH₂)₁₄ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is ##STR56## or (f)R'₁ and R'₂ are --CH₂ (CH₂)₂ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is ##STR57##or (g) R'₁ R'₂ are --CH₂ (CH₂)₁₄ CH₃, R'₃ is --CH₂ CH₃, and R'₄ is##STR58##
 3. A compound of the formula: ##STR59## wherein R"₁, R"₂, R"₃and R"₄ are all --CH₂ (CH₂)₁₄ CH₃.
 4. A compound of the formula:##STR60## wherein: (a) R"₇ and R"₈ are --CH₂ (CH₂)₁₄ CH₃ ;(b) R"₇ ishydrogen and R"₈ is --CH₃ ; (c) R"₇ is hydrogen and R₈ is ##STR61## (d)R"₇ is hydrogen and R"₈ is ##STR62##